Compounds for enzyme inhibition

ABSTRACT

Peptide-based compounds including heteroatom-containing, three-membered rings efficiently and selectively inhibit specific activities of N-terminal nucleophile (Ntn) hydrolases. The activities of those Ntn having multiple activities can be differentially inhibited by the compounds described. For example, the chymotrypsin-like activity of the 20S proteasome may be selectively inhibited with the inventive compounds. The peptide-based compounds include at least three peptide units, an epoxide or aziridine, and functionalization at the N-terminus. Among other therapeutic utilities, the peptide-based compounds are expected to display anti-inflammatory properties and inhibition of cell proliferation.

CLAIM OF PRIORITY

This application is a continuation of United States (“U.S.”) applicationSer. No. 11/578,626, which has a mail date of Oct. 16, 2006 and a 35U.S.C. §371(c) date of Aug. 10, 2007 and is the U.S. National Stage ofInternational Application No. PCT/US2005/012740, filed on Apr. 14, 2005,which in turn claims the benefit of U.S. Provisional Application No.60/562,340, filed on Apr. 15, 2004; U.S. Provisional Application No.60/569,096, filed on May 7, 2004; U.S. Provisional Application No.60/599,401, filed on Aug. 6, 2004; U.S. Provisional Application No.60/610,001, filed on Sep. 14, 2004; U.S. Provisional Application No.60/610,002, filed on Sep. 14, 2004; U.S. Provisional Application No.60/610,159, filed on Sep. 14, 2004; and U.S. Provisional Application No.60/620,573, filed on Oct. 20, 2004; each of these prior filedapplications is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to compounds and methods for enzyme inhibition.In particular, the invention relates to therapeutic methods based onenzyme inhibition.

BACKGROUND OF THE INVENTION

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I presentation,apoptosis, cell division, and NF-κB activation.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings thatplays important roles in cell growth regulation, majorhistocompatibility complex class I presentation, apoptosis, antigenprocessing, NF-κB activation, and transduction of pro-inflammatorysignals. In yeast and other eukaryotes, 7 different α subunits form theouter rings and 7 different β subunits comprise the inner rings. The αsubunits serve as binding sites for the 19S (PA700) and 11S (PA28)regulatory complexes, as well as a physical barrier for the innerproteolytic chamber formed by the two β subunit rings. Thus, in vivo,the proteasome is believed to exist as a 26S particle (“the 26Sproteasome”). In vivo experiments have shown that inhibition of the 20Sform of the proteasome can be readily correlated to inhibition of 26Sproteasome. Cleavage of amino-terminal prosequences of β subunits duringparticle formation expose amino-terminal threonine residues, which serveas the catalytic nucleophiles. The subunits responsible for catalyticactivity in proteasome thus possess an amino terminal nucleophilicresidue, and these subunits belong to the family of N-terminalnucleophile (Ntn) hydrolases (where the nucleophilic N-terminal residueis, for example, Cys, Ser, Thr, and other nucleophilic moieties). Thisfamily includes, for example, penicillin G acylase (PGA), penicillin Vacylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three γ-interferon-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, X, Y and Z respectively, thus altering the catalyticactivities of the proteasome. Through the use of different peptidesubstrates, three major proteolytic activities have been defined for theeukaryote 20S proteasome: chymotrypsin-like activity (CT-L), whichcleaves after large hydrophobic residues; trypsin-like activity (T-L),which cleaves after basic residues; and peptidylglutamyl peptidehydrolyzing activity (PGPH), which cleaves after acidic residues. Twoadditional less characterized activities have also been ascribed to theproteasome: BrAAP activity, which cleaves after branched-chain aminoacids; and SNAAP activity, which cleaves after small neutral aminoacids. The major proteasome proteolytic activities appear to becontributed by different catalytic sites, since inhibitors, pointmutations in β subunits and the exchange of γ interferon-inducing βsubunits alter these activities to various degrees.

There are several examples of small molecules which have been used toinhibit proteasome activity; however, these compounds generally lack thespecificity, stability, or potency necessary to explore and exploit theroles of the proteasome at the cellular and molecular level. Therefore,the synthesis of small molecule inhibitor(s) with increased sitespecificity, improved stability and solubility, and increased potencyare needed to allow the exploration of the roles of the proteasome atthe cellular and molecular level.

SUMMARY OF THE INVENTION

The invention relates to analogs and prodrugs of classes of moleculesknown as peptide α′,β′-epoxides and peptide α′,β′-aziridines. The parentmolecules are understood to bind efficiently, irreversibly andselectively to N-terminal nucleophile (Ntn) hydrolases, and canspecifically inhibit particular activities of enzymes having multiplecatalytic activity.

Once thought merely to dispose of denatured and misfolded proteins, theproteasome is now recognized as constituting proteolytic machinery thatregulates the levels of diverse intracellular proteins through theirdegradation in a signal-dependent manner. Hence, there is great interestin identifying reagents that can specifically perturb the activities ofthe proteasome and other Ntn hydrolases and thereby be used as probes tostudy the role of these enzymes in biological processes. Analogs andprodrugs for compounds that target the Ntn hydrolases are hereindescribed, synthesized, and investigated. Peptide epoxides and peptideaziridines that can potently, selectively, and irreversibly inhibitparticular proteasome activities are disclosed and claimed.

Unlike several other peptide-based inhibitors, the peptide epoxides andpeptide aziridines described herein are not expected to substantiallyinhibit non-proteasomal proteases such as trypsin, chymotrypsin,cathepsin B, papain, and calpain at concentrations up to 50 μM. Athigher concentrations, inhibition may be observed, but would be expectedto be competitive and not irreversible, if the inhibitor merely competeswith the substrate. The novel peptide epoxides and peptide aziridinesare also expected to inhibit NF-κB activation and to stabilize p53levels in cell culture. Moreover, these compounds would be expected tohave anti-inflammatory activity. Thus, these compounds can be uniquemolecular probes, which have the versatility to explore Ntn enzymefunction in normal biological and pathological processes.

In one aspect, the invention provides inhibitor analogs and prodrugscomprising a heteroatom-containing three-membered ring. These inhibitorscan inhibit catalytic activity of N-terminal nucleophile hydrolaseenzymes (for example, the 20S proteasome, or the 26S proteasome) whensaid inhibitor is present at concentrations below about 50 μM. Regardingthe 20S proteasome, particular hydrolase inhibitors inhibitchymotrypsin-like activity of the 20S proteasome when the inhibitor ispresent at concentrations below about 5 μM, and does not inhibittrypsin-like activity or PGPH activity of the 20S proteasome whenpresent at concentrations below about 5 μM. The hydrolase inhibitor maybe, for example, a peptide α′,β′-epoxy ketone or α′,β′-aziridine ketone,and the peptide may be a tetrapeptide. The tetrapeptide may includebranched or unbranched side chains such as hydrogen, C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, C₁₋₆aralkyl, C₁₋₆alkylamide,C₁₋₆alkylamine, C₁₋₆-carboxylic acid, C₁₋₆-carboxyl ester,C₁₋₆alkylthiol, or C₁₋₆alkylthioether, for example isobutyl, 1-naphthyl,phenylmethyl, and 2-phenylethyl. The α′-carbon of the α′,β′-epoxy ketoneor α′,β′-aziridine ketone may be a chiral carbon atom, such as an (R) orβ configured carbon, as these are defined herein.

In another aspect, the invention provides pharmaceutical compositions,including a pharmaceutically acceptable carrier and a pharmaceuticallyeffective amount of the hydrolase inhibitor analog or prodrug, whichameliorates the effects of neurodegenerative disease (such asAlzheimer's disease), muscle-wasting diseases, cancer, chronicinfectious diseases, fever, muscle disuse, denervation, nerve injury,fasting, and immune-related conditions, among others.

In another aspect, the invention provides anti-inflammatorycompositions.

In another aspect, the invention provides methods for the following:inhibiting or reducing HIV infection in a subject; affecting the levelof viral gene expression in a subject; altering the variety of antigenicpeptides produced by the proteasome in an organism; determining whethera cellular, developmental, or physiological process or output in anorganism is regulated by the proteolytic activity of a particular Ntnhydrolase; treating Alzheimer's disease in a subject; reducing the rateof muscle protein degradation in a cell; reducing the rate ofintracellular protein degradation in a cell; reducing the rate of p53protein degradation in a cell; inhibiting the growth of p53-relatedcancers in a subject; inhibiting antigen presentation in a cell;suppressing the immune system of a subject; inhibiting IκB-α degradationin an organism; reducing the content of NF-κB in a cell, muscle, organor subject; affecting cyclin-dependent eukaryotic cell cycles; treatingproliferative disease in a subject; affecting proteasome-dependentregulation of oncoproteins in a cell; treating cancer growth in asubject; treating p53-related apoptosis in a subject; and screeningproteins processed by N-terminal nucleophile hydrolases in a cell. Eachof these methods involves administering or contacting an effectiveamount of a composition comprising the hydrolase inhibitors disclosedherein, to a subject, a cell, a tissue, an organ, or an organism.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves compounds useful as enzyme inhibitors. Thesecompounds are generally useful to inhibit enzymes having a nucleophilicgroup at the N-terminus For example, activities of enzymes or enzymesubunits having N-terminal amino acids with nucleophiles in their sidechains, such as threonine, serine, or cysteine can be successfullyinhibited by the enzyme inhibitors described herein. Activities ofenzymes or enzyme subunits having non-amino acid nucleophilic groups attheir N-termini, such as, for example, protecting groups orcarbohydrates, can also be successfully inhibited by the enzymeinhibitors described herein.

While not bound by any particular theory of operation, it is believedthat such N-terminal nucleophiles of Ntn form covalent adducts with theepoxide functional group of the enzyme inhibitors described herein. Forexample, in the β5/Pre2 subunit of 20S proteasome, the N-terminalthreonine is believed to irreversibly form a morpholino or piperazinoadduct upon reaction with a peptide epoxide or aziridine such as thosedescribed below. Such adduct formation would involve ring-openingcleavage of the epoxide or aziridine.

In embodiments including such groups bonded to α′ carbons, thestereochemistry of the α′-carbon (that carbon forming a part of theepoxide or aziridine ring) can be (R) or (S). The invention is based, inpart, on the structure-function information disclosed herein, whichsuggests the following preferred stereochemical relationships. Note thata preferred compound may have a number of stereocenters having theindicated up-down (or β-α, where β as drawn herein is above the plane ofthe page) or (R)—(S) relationship (that is, it is not required thatevery stereocenter in the compound conform to the preferences stated).In some preferred embodiments, the stereochemistry of the α′ carbon is(R), that is, the X atom is p, or above the plane of the molecule.

Regarding the stereochemistry, the Cahn-Ingold-Prelog rules fordetermining absolute stereochemistry are followed. These rules aredescribed, for example, in Organic Chemistry, Fox and Whitesell; Jonesand Bartlett Publishers, Boston, Mass. (1994); Section 5-6, pp 177-178,which section is hereby incorporated by reference. Peptides can have arepeating backbone structure with side chains extending from thebackbone units. Generally, each backbone unit has a side chainassociated with it, although in some cases, the side chain is a hydrogenatom. In other embodiments, not every backbone unit has an associatedside chain. Peptides useful in peptide epoxides or peptide aziridineshave two or more backbone units. In some embodiments useful forinhibiting chymotrypsin-like (CT-L) activity of the proteasome, betweenfour and eight backbone units are present, and in some preferredembodiments for CT-L inhibition, between four and six backbone units arepresent.

The side chains extending from the backbone units can include naturalaliphatic or aromatic amino acid side chains, such as hydrogen(glycine), methyl (alanine), isopropyl (valine), sec-butyl (isoleucine),isobutyl (leucine), phenylmethyl (phenylalanine), and the side chainconstituting the amino acid proline. The side chains can also be otherbranched or unbranched aliphatic or aromatic groups such as ethyl,n-propyl, n-butyl, t-butyl, and aryl substituted derivatives such as1-phenylethyl, 2-phenylethyl, (1-naphthyl)methyl, (2-naphthyl)methyl,1-(1-naphthyl)ethyl, 1-(2-naphthyl)ethyl, 2-(1-naphthyl)ethyl,2-(2-naphthyl)ethyl, and similar compounds. The aryl groups can befurther substituted with branched or unbranched C₁₋₆alkyl groups, orsubstituted alkyl groups, acetyl and the like, or further aryl groups,or substituted aryl groups, such as benzoyl and the like. Heteroarylgroups can also be used as side chain substituents. Heteroaryl groupsinclude nitrogen-, oxygen-, and sulfur-containing aryl groups such asthienyl, benzothienyl, naphthothienyl, thianthrenyl, furyl, pyranyl,isobenzofuranyl, chromenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl,pyrazinyl, indolyl, purinyl, quinolyl, and the like.

In some embodiments, polar or charged residues can be introduced intothe peptide epoxides or peptide aziridines. For example, naturallyoccurring amino acids such as hydroxy-containing (Thr, Tyr, Ser) orsulfur-containing (Met, Cys) can be introduced, as well as non-essentialamino acids, for example, taurine, carnitine, citrulline, cystine,ornithine, norleucine and others. Non-naturally occurring side chainsubstituents with charged or polar moieties can also be included, suchas, for example, C₁₋₆alkyl chains or C₆₋₁₂aryl groups with one or morehydroxy, short chain alkoxy, sulfide, thio, carboxyl, ester, phospho,amido or amino groups, or such substituents substituted with one or morehalogen atoms. In some preferred embodiments, there is at least one arylgroup present in a side chain of the peptide moiety.

In some embodiments, the backbone units are amide units[—NH—CHR—C(═O)—], in which R is the side chain. Such a designation doesnot exclude the naturally occurring amino acid proline, or othernon-naturally occurring cyclic secondary amino acids, which will berecognized by those of skill in the art.

In other embodiments, the backbone units are N-alkylated amide units(for example, N-methyl and the like), olefinic analogs (in which one ormore amide bonds are replaced by olefinic bonds), tetrazole analogs (inwhich a tetrazole ring imposes a cis-configuration on the backbone), orcombinations of such backbone linkages. In still other embodiments, theamino acid α-carbon is modified by α-alkyl substitution, for example,aminoisobutyric acid. In some further embodiments, side chains arelocally modified, for example, by Δ^(E) or Δ^(Z) dehydro modification,in which a double bond is present between the α and β atoms of the sidechain, or for example by Δ^(E) or Δ^(Z) cyclopropyl modification, inwhich a cyclopropyl group is present between the a and β atoms of theside chain. In still further embodiments employing amino acid groups,D-amino acids can be used. Further embodiments can include sidechain-to-backbone cyclization, disulfide bond formation, lactamformation, azo linkage, and other modifications discussed in “Peptidesand Mimics, Design of Conformationally Constrained” by Hruby and Boteju,in “Molecular Biology and Biotechnology: A Comprehensive DeskReference”, ed. Robert A. Meyers, VCH Publishers (1995), pp. 658-664,which is hereby incorporated by reference.

In certain embodiments, the subject compounds are analogs or prodrugs ofthe compounds disclosed in U.S. application Ser. No. 09/569,748, thecontents of which are hereby incorporated by reference in theirentirety. Suitable enzyme inhibitor analogs or prodrugs may have astructure of formula (I) or a pharmaceutically acceptable salt thereof,

-   wherein each A is independently selected from C═O, C═S, and SO₂,    preferably C═O; or-   A is optionally a covalent bond when adjacent to an occurrence of Z;-   L is absent or is selected from C═O, C═S, and SO₂, preferably L is    absent or C═O;-   M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;-   Q is absent or is selected from O, NH, and N—C₁₋₆alkyl, preferably Q    is absent, O, or NH, most preferably Q is absent or O;-   X is selected from O, NH, and N—C₁₋₆alkyl, preferably O;-   Y is absent or is selected from O, NH, N—C₁₋₆alkyl, S, SO, SO₂,    CHOR¹⁰, and CHCO₂R¹⁰;-   each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,    preferably O; or-   Z is optionally a covalent bond when adjacent to an occurrence of A;-   R¹, R², R³, and R⁴ are each independently selected from C₁₋₆alkyl,    C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of    which is optionally substituted with one or more of amide, amine,    carboxylic acid (or a salt thereof), ester (including C₁₋₅ alkyl    ester and aryl ester), thiol, or thioether substituents;-   R⁵ is N(R⁶)LQR⁷;    R⁶, R¹², R¹³, and R¹⁴ are independently selected from hydrogen, OH,    C₁₋₆alkyl, and a group of formula IV; preferably, R⁶ is selected    from hydrogen, OH, and C₁₋₆alkyl, and R¹², R¹³, and R¹⁴ are    independently selected from hydrogen and C₁₋₆alkyl, preferably    hydrogen;

-   R⁷ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,    aryl, C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R⁸ZAZ—C₁₋₈alkyl-,    R¹¹Z—C₁₋₈alkyl-, (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    R⁸ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-, (R¹⁰)₂N—C₁₋₁₂alkyl-,    (R¹⁰)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-,    R¹¹SO₂C₁₋₈alkyl-, and R¹¹SO₂NH; preferably C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl,    R⁸ZA-C₁₋₈alkyl-, R¹¹Z—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,    R⁸ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-, (R¹⁶)₂N—C₁₋₈alkyl-,    (R¹⁰)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,    R¹¹SO₂C₁₋₈alkyl-, and R¹¹SO₂NH, wherein each occurrence of Z and A    is independently other than a covalent bond; or-   R⁶ and R⁷ together are C₁₋₆alkyl-Y—C₁₋₆alkyl,    C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,    ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, thereby forming a ring;    preferably C₁₋₂alkyl-Y—C₁₋₂alkyl, C₁₋₂alkyl-ZA-C₁₋₂alkyl,    A-C₁₋₂alkyl-ZA-C₁₋₂alkyl, A-C₁₋₃alkyl-A, or C₁₋₄alkyl-A, wherein    each occurrence of Z and A is independently other than a covalent    bond;-   R⁸ and R⁹ are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl,    and C₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, and    C₁₋₆alkyl, or R⁸ and R⁹ together are C₁₋₆alkyl, thereby forming a    ring;-   each R¹⁰ is independently selected from hydrogen and C₁₋₆alkyl,    preferably C₁₋₆alkyl; and-   R¹¹ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl,    C₁₋₆aralkyl, and C₁₋₆heteroaralkyl,    -   R¹⁵ and R¹⁰ are independently selected from hydrogen and        C₁₋₆alkyl, or R¹⁵ and R¹⁰ together form a 3- to 6-membered        carbocyclic or heterocyclic ring; and    -   R¹⁷ and R¹⁸ are independently selected from hydrogen, a metal        cation, C₁₋₆alkyl, and C₁₋₆aralkyl, or R¹⁷ and R¹⁸ together        represent C₁₋₆alkyl, thereby forming a ring;-   provided that when R⁶, R¹², R¹³, and R¹⁴ are H or CH₃, and Q is    absent, LR⁷ is not hydrogen, unsubstituted C₁₋₆alkylC═O, a further    chain of amino acids, t-butoxycarbonyl (Boc), benzoyl (Bz),    fluoren-9-ylmethoxycarbonyl (Fmoc), triphenylmethyl(trityl),    benzyloxycarbonyl (Cbz), trichloroethoxycarbonyl (Troc); or    substituted or unsubstituted aryl or heteroaryl; and-   in any occurrence of the sequence ZAZ, at least one member of the    sequence must be other than a covalent bond.

In certain embodiments, when R⁶ is H, L is C═O, and Q is absent, R⁷ isnot hydrogen, C₁₋₆alkyl, or substituted or unsubstituted aryl orheteroaryl. In certain embodiments, when R⁶ is H and Q is absent, R⁷ isnot a protecting group such as those described in Greene, T. W. andWuts, P. G. M., “Protective Groups in Organic Synthesis”, John Wiley &Sons, 1999 or Kocieński, P. J., “Protecting Groups”, Georg ThiemeVerlag, 1994.

In some embodiments, R¹, R², R³, and R⁴ are selected from C₁₋₆alkyl orC₁₋₆aralkyl. In preferred embodiments, R² and R⁴ are C₁₋₆alkyl and R¹and R³ are C₁₋₆aralkyl. In the most preferred embodiment, R² and R⁴ areisobutyl, R¹ is 2-phenylethyl, and R³ is phenylmethyl.

In certain embodiments, L and Q are absent and R⁷ is selected fromC₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, C₁₋₆aralkyl, and C₁₋₆heteroaralkyl.In certain such embodiments, R⁶ is C₁₋₆alkyl and R⁷ is selected frombutyl, allyl, propargyl, phenylmethyl, 2-pyridyl, 3-pyridyl, and4-pyridyl.

In other embodiments, L is SO₂, Q is absent, and R⁷ is selected fromC₁₋₆alkyl and aryl. In certain such embodiments, R⁷ is selected frommethyl and phenyl.

In certain embodiments, L is C═O and R⁷ is selected from C₁₋₆alkyl,C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl,C₁₋₆heteroaralkyl, R⁸ZA-C₁₋₈alkyl-, R¹¹Z—C₁₋₈alkyl-,(R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-, (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, R⁸ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,heterocyclylMZAZ—C₁₋₈alkyl-, (R¹⁰)₂N—C₁₋₈alkyl-, (R¹⁰)₃N⁺—C₁₋₈alkyl-,heterocyclylM-, carbocyclylM-, R¹¹SO₂C₁₋₈alkyl-, and R¹¹SO₂NH—, whereineach occurrence of Z and A is independently other than a covalent bond.In certain embodiments, L is C═O, Q is absent, and R⁷ is H.

In certain embodiments, R⁶ is C₁₋₆alkyl, R⁷ is C₁₋₆alkyl, Q is absent,and L is C═O. In certain such embodiments, R⁷ is ethyl, isopropyl,2,2,2-trifluoroethyl, or 2-(methylsulfonyl)ethyl.

In other embodiments, L is C═O, Q is absent, and R⁷ is C₁₋₆aralkyl. Incertain such embodiments, R⁷ is selected from 2-phenylethyl,phenylmethyl, (4-methoxyphenyl)methyl, (4-chlorophenyl)methyl, and(4-fluorophenyl)methyl.

In other embodiments, L is C═O, Q is absent, R⁶ is C₁₋₆alkyl, and R⁷ isaryl. In certain such embodiments, R⁷ is substituted or unsubstitutedphenyl.

In certain embodiments, L is C═O, Q is absent or O, n is 0 or 1, and R⁷is —(CH₂)_(n)carbocyclyl. In certain such embodiments, R⁷ is cyclopropylor cyclohexyl.

In certain embodiments, L and A are C═O, Q is absent, Z is O, n is aninteger from 1 to 8 (preferably 1), and R⁷ is selected fromR⁸ZA-C₁₋₈alkyl-, R¹¹Z—C₁₋₈alkyl-, R⁸ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,(R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-, andheterocyclylMZAZ—C₁₋₈alkyl-, wherein each occurrence of A isindependently other than a covalent bond. In certain such embodiments,R⁷ is heterocyclylMZAZ—C₁₋₈alkyl- where heterocyclyl is substituted orunsubstituted oxodioxolenyl or N(R¹²)(R¹³), wherein R¹² and R¹³ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, preferably C₁₋₃alkyl-Y—C₁₋₃ alkyl, therebyforming a ring.

In certain preferred embodiments, L is C═O, Q is absent, n is an integerfrom 1 to 8, and R⁷ is selected from (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-,(R¹⁰)₂NC₁₋₈alkyl, (R¹⁰)₃N⁺(CH₂)_(n)—, and heterocyclyl-M-. In certainsuch embodiments, R⁷ is —C₁₋₈alkylN(R¹⁰)₂ or —C₁₋₈alkylN⁺(R¹⁰)₃, whereR¹⁰ is C₁₋₆alkyl. In certain other such embodiments, R⁷ isheterocyclylM-, where heterocyclyl is selected from morpholino,piperidino, piperazino, and pyrrolidino.

In certain embodiments, L is C═O, R⁶ is C₁₋₆alkyl, Q is selected from Oand NH and R⁷ is selected from C₁₋₆alkyl, cycloalkyl-M, C₁₋₆aralkyl, andC₁₋₆heteroaralkyl. In other embodiments, L is C═O, R⁶ is C₁₋₆alkyl, Q isselected from O and NH, and R⁷ is C₁₋₆alkyl, where C₁₋₆alkyl is selectedfrom methyl, ethyl, and isopropyl. In further embodiments, L is C═O, R⁶is C₁₋₆alkyl, Q is selected from O and NH and R⁷ is C₁₋₆aralkyl, wherearalkyl is phenylmethyl. In other embodiments, L is C═O, R⁶ isC₁₋₆alkyl, Q is selected from O and NH, and R⁷ is C₁₋₆heteroaralkyl,where heteroaralkyl is (4-pyridyl)methyl.

In certain embodiments, L is absent or is C═O, and R⁶ and R⁷ togetherare C₁₋₆alkyl-Y—C₁₋₆alkyl, C₁₋₆alkyl-ZA-C₁₋₆alkyl, or C₁₋₆alkyl-A,wherein each occurrence of Z and A is independently other than acovalent bond, thereby forming a ring. In certain preferred embodiments,L is C═O, Q and Y are absent, and R⁶ and R⁷ together areC₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, L and Q areabsent, and R⁶ and R⁷ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In anotherpreferred embodiment, L is C═O, Q is absent, Y is selected from NH andN—C₁₋₆alkyl, and R⁶ and R⁷ together are C₁₋₃alkyl-Y—C₁₋₃alkyl. Inanother preferred embodiment, L is C═O, Y is absent, and R⁶ and R⁷together are C₁₋₃alkyl-Y—C₁₋₃alkyl. In another preferred embodiment, Land A are C═O, and R⁶ and R⁷ together are C₁₋₂alkyl-ZA-C₁₋₂alkyl. Inanother preferred embodiment, L and A are C═O and R⁶ and R⁷ together areC₂₋₃alkyl-A.

In certain embodiments, a compound of formula I has the followingstereochemistry:

In preferred embodiments, the inhibitor has a structure of formula II ora pharmaceutically acceptable salt thereof,

-   wherein each A is independently selected from C═O, C═S, and SO₂,    preferably C═O; or-   A is optionally a covalent bond when adjacent to an occurrence of Z;-   L is absent or is selected from C═O, C═S, and SO₂, preferably L is    absent or C═O;-   M is absent or is C₁₋₁₂alkyl, preferably C₁₋₈alkyl;-   Q is absent or is selected from O, NH, and N—C₁₋₆alkyl, preferably Q    is absent, O, or NH, most preferably Q is absent or O;-   X is selected from O, NH, and N—C₁₋₆alkyl, preferably O;-   Y is absent or is selected from O, NH, N—C₁₋₆alkyl, S, SO, SO₂,    CHOR¹⁰, and CHCO₂R¹⁰;-   each Z is independently selected from O, S, NH, and N—C₁₋₆alkyl,    preferably O; or-   Z is optionally a covalent bond when adjacent to an occurrence of A;-   R² and R⁴ are each independently selected from C₁₋₆alkyl,    C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, any of    which is optionally substituted with one or more of amide, amine,    carboxylic acid (or a salt thereof), ester (including C₁₋₅ alkyl    ester and aryl ester), thiol, or thioether substituents;-   R⁵ is N(R⁶)LQR⁷;-   R⁶ is selected from hydrogen, OH, and C₁₋₆alkyl, preferably    C₁₋₆alkyl;-   R⁷ is selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl,    aryl, C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl, R⁸ZAZ—C₁₋₈alkyl-,    R¹¹Z—C₁₋₈alkyl-, (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    R⁸ZAZ—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-, (R¹⁰)₂N—C₁₋₁₂alkyl-,    (R¹⁰)₃N⁺—C₁₋₁₂alkyl-, heterocyclylM-, carbocyclylM-,    R¹¹SO₂C₁₋₈alkyl-, and R¹¹SO₂NH; preferably C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, aryl, C₁₋₆aralkyl, heteroaryl, C₁₋₆heteroaralkyl,    R⁸ZA-C₁₋₈alkyl-, R¹¹Z—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-Z—C₁₋₈alkyl-,    R⁸ZA-C₁₋₈alkyl-ZAZ—C₁₋₈alkyl-, heterocyclylMZAZ—C₁₋₈alkyl-,    (R⁸O)(R⁹O)P(═O)O—C₁₋₈alkyl-, (R¹⁰)₂N—C₁₋₈alkyl-,    (R¹⁰)₃N⁺—C₁₋₈alkyl-, heterocyclylM-, carbocyclylM-,    R¹¹SO₂C₁₋₈alkyl-, and R¹¹SO₂NH, wherein each occurrence of Z and A    is independently other than a covalent bond; or-   R⁶ and R⁷ together are C₁₋₆alkyl-Y—C₁₋₆alkyl,    C₁₋₆alkyl-ZAZ—C₁₋₆alkyl, ZAZ—C₁₋₆alkyl-ZAZ—C₁₋₆alkyl,    ZAZ—C₁₋₆alkyl-ZAZ, or C₁₋₆alkyl-A, thereby forming a ring;    preferably C₁₋₂alkyl-Y—C₁₋₂alkyl, C₁₋₂alkyl-ZA-C₁₋₂alkyl,    A-C₁₋₂alkyl-ZA-C₁₋₂alkyl, A-C₁₋₃alkyl-A, or C₁₋₄alkyl-A, wherein    each occurrence of Z and A is independently other than a covalent    bond;-   R⁸ and R⁹ are independently selected from hydrogen, metal cation,    C₁₋₆alkyl, C₁₋₆alkenyl, C₁₋₆alkynyl, aryl, heteroaryl, C₁₋₆aralkyl,    and C₁₋₆heteroaralkyl, preferably from hydrogen, metal cation, and    C₁₋₆alkyl, or R⁸ and R⁹ together are C₁₋₆alkyl, thereby forming a    ring;-   each R¹⁰ is independently selected from hydrogen and C₁₋₆alkyl,    preferably C₁₋₆alkyl; and-   R¹¹ is independently selected from hydrogen, C₁₋₆alkyl, C₁₋₆alkenyl,    C₁₋₆alkynyl, carbocyclyl, heterocyclyl, aryl, heteroaryl,    C₁₋₆aralkyl, and C₁₋₆heteroaralkyl,-   provided that when R⁶ is H or CH₃ and Q is absent, LR⁷ is not    hydrogen, unsubstituted C₁₋₆alkylC═O, a further chain of amino    acids, t-butoxycarbonyl (Boc), benzoyl (Bz),    fluoren-9-ylmethoxycarbonyl (Fmoc), triphenylmethyl(trityl),    benzyloxycarbonyl (Cbz), trichloroethoxycarbonyl (Troc); or    substituted or unsubstituted aryl or heteroaryl; and-   in any occurrence of the sequence ZAZ, at least one member of the    sequence must be other than a covalent bond.

In certain embodiments, L is C═O, Q is absent, X is O, R⁶ is H, and R²and R⁴ are selected from C₁₋₆alkyl and C₁₋₆aralkyl. In preferred suchembodiments, R² and R⁴ are C₁₋₆alkyl. In the most preferred suchembodiment, R² and R⁴ are isobutyl.

In certain embodiments, L is C═O, Q is absent, X is O, R⁶ is H, R² andR⁴ are isobutyl, and R⁷ is heterocyclylM-, where the heterocycle is anitrogen-containing heterocycle, such as piperazino (including N-(loweralkyl) piperazino), morpholino, and piperidino. In preferred suchembodiments, M is CH₂.

In certain embodiments, a compound of formula II is selected from:

In certain embodiments, the subject compounds are phosphate-containingprodrugs of the compounds disclosed in U.S. application Ser. No.09/569,748, the contents of which are hereby incorporated by referencein their entirety. The enzyme inhibitor prodrugs for inhibition ofchymotrypsin-like (CT-L) activity of Ntn have a structure of formula(III) or a pharmaceutically acceptable salt thereof

whereinX is selected from O, NH, and N-alkyl, preferably O;R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula IV, with the proviso that at least one of R¹, R², R³, and R⁴is a group of formula IV;

R⁵, R⁶, R⁷, and R⁸ are independently selected from C₁₋₆alkyl,C₁₋₆hydroxyalkyl, C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of whichis optionally substituted with a group selected from amide, amine,carboxylic acid or a pharmaceutically acceptable salt thereof, carboxylester, thiol, and thioether;

R⁹ is a further chain of amino acids, hydrogen, C₁₋₆acyl, a protectinggroup, aryl, or heteroaryl, where substituents may include halogen,carbonyl, nitro, hydroxy, aryl, and C₁₋₅alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C₁₋₆alkyl, orR¹⁰ and R¹¹ together form a 3- to 6-membered carbocyclic or heterocyclicring;

R¹² and R¹³ are independently selected from hydrogen, a metal cation,C₁₋₆alkyl, and C₁₋₆aralkyl, or R¹² and R¹³ together represent C₁₋₆alkyl,thereby forming a ring; and

L is absent or is selected from —CO₂ or —C(═S)O.

Suitable N-terminal protecting groups known in the art of peptidesyntheses, include t-butoxy carbonyl (Boc), benzoyl (Bz),fluoren-9-ylmethoxycarbonyl (Fmoc), triphenylmethyl (trityl) andtrichloroethoxycarbonyl (Troc) and the like. The use of variousN-protecting groups, e.g., the benzyloxy carbonyl group or thet-butyloxycarbonyl group (Boc), various coupling reagents, e.g.,dicyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC),N-hydroxyazabenzotriazole (HATU), carbonyldiimidazole, or1-hydroxybenzotriazole monohydrate (HOBT), and various cleavageconditions: for example, trifluoracetic acid (TFA), HCl in dioxane,hydrogenation on Pd—C in organic solvents (such as methanol or ethylacetate), boron tris(trifluoroacetate), and cyanogen bromide, andreaction in solution with isolation and purification of intermediatesare well-known in the art of peptide synthesis, and are equallyapplicable to the preparation of the subject compounds.

In some embodiments, any two of R¹, R², R³, and R⁴ are hydrogen and anytwo of R¹, R², R³, and R⁴ have a structure of formula IV. In preferredembodiments any three of R¹, R², R³, and R⁴ are hydrogen and any one ofR¹, R², R³, and R⁴ has a structure of formula IV. In the most preferredembodiment, R¹ has a structure of formula IV and R², R³, and R⁴ arehydrogen.

In certain embodiments, R⁵, R⁶, R⁷, and R⁸ are C₁₋₆alkyl or C₁₋₆aralkyl.In preferred embodiments, R⁶ and R⁸ are C₁₋₆alkyl and R⁵ and R⁷ areC₁₋₆aralkyl. In the most preferred embodiment, R⁶ and R⁸ are isobutyl,R⁵ is 2-phenylethyl, and R⁷ is phenylmethyl. In certain embodiments, R⁹is selected from hydrogen, C₁₋₆acyl, or a protecting group. In preferredembodiments, R⁹ is hydrogen or acetyl. In the most preferred embodiment,R⁹ is acetyl.

In certain embodiments, R¹⁰ and R¹¹ are selected from hydrogen andC₁₋₆alkyl. In a preferred embodiment, R¹⁰ is hydrogen and R¹¹ isC₁₋₆alkyl. In a further preferred embodiment, R¹⁰ is hydrogen and R¹¹ ismethyl. In another preferred embodiment, both R¹⁰ and R¹¹ are hydrogen.In certain embodiments, R¹² and R¹³ are C₁₋₆alkyl, metal cation, orC₁₋₆aralkyl. In certain preferred embodiments, R¹² and R¹³ are selectedfrom benzyl, tert-butyl, and sodium cation. In more preferredembodiments, both R¹² and R¹³ are benzyl or tert-butyl. In the mostpreferred embodiment, at least one of R¹² and R¹³ is a sodium cation.

In certain embodiments, a compound of formula III has the followingstereochemistry:

In preferred embodiments, the inhibitor has a structure of formula V ora pharmaceutically acceptable salt thereof,

wherein

X is selected from O, NH, and N-alkyl, preferably O;

R¹, R², R³, and R⁴ are independently selected from hydrogen and a groupof formula IV, with the proviso that at least one of R¹, R², R³, and R⁴is a group of formula IV;

R⁶ and R⁸ are independently selected from C₁₋₆alkyl, C₁₋₆hydroxy alkyl,C₁₋₆alkoxyalkyl, aryl, and C₁₋₆aralkyl, each of which is optionallysubstituted with a group selected from amide, amine, carboxylic acid ora pharmaceutically acceptable salt thereof, carboxyl ester, thiol, andthioether;

R⁹ is a further chain of amino acids, hydrogen, acyl, a protectinggroup, aryl, or heteroaryl, where substituents may include halogen,carbonyl, nitro, hydroxy, aryl, and C₁₋₅alkyl. Suitable N-terminalprotecting groups known in the art of peptide syntheses, includet-butoxy carbonyl (Boc), benzoyl (Bz), fluoren-9-ylmethoxycarbonyl(Fmoc), triphenylmethyl (trityl) and trichloroethoxycarbonyl (Troc) andthe like; and

In some embodiments, any two of R¹, R², R³, and R⁴ are hydrogen and anytwo of R¹, R², R³, and R⁴ have a structure of formula IV. In preferredembodiments any three of R¹, R², R³, and R⁴ are hydrogen and any one ofR¹, R², R³, and R⁴ has a structure of formula IV. In the most preferredembodiment, R¹ has a structure of formula IV and R², R³, and R⁴ arehydrogen.

In certain embodiments, R⁶ and R⁸ are C₁₋₆alkyl or C₁₋₆aralkyl. Inpreferred embodiments, R⁶ and R⁸ are C₁₋₆alkyl. In the most preferredembodiment, R⁶ and R⁸ are isobutyl. In certain embodiments, R⁹ isselected from hydrogen, C₁₋₆acyl, or a protecting group. In preferredembodiments, R⁹ is hydrogen or acetyl. In the most preferred embodiment,R⁹ is acetyl.

In certain embodiments, R¹⁰ and R¹¹ are selected from hydrogen andC₁₋₆alkyl. In a preferred embodiment, R¹⁰ is hydrogen and R¹¹ isC₁₋₆alkyl. In a further preferred embodiment, R¹⁰ is hydrogen and R¹¹ ismethyl. In another preferred embodiment, both R¹⁰ and R¹¹ are hydrogen.In certain embodiments, R¹² and R¹³ are C₁₋₆alkyl, metal cation, orC₁₋₆aralkyl. In certain preferred embodiments, R¹² and R¹³ are selectedfrom benzyl, tert-butyl, and sodium cation. In more preferredembodiments, both R¹² and R¹³ are benzyl or tert-butyl. In the mostpreferred embodiment, at least one of R¹² and R¹³ is a sodium cation.

In certain embodiments, R⁶ and R⁸ are C₁₋₆alkyl. In preferredembodiments, R⁶ and R⁸ are isobutyl. In preferred embodiments, R⁹ ishydrogen or acetyl. In the most preferred embodiments, R⁹ is acetyl. Ina preferred embodiment, R¹⁰ is hydrogen and R¹¹ is methyl. In anotherpreferred embodiment, both R¹⁰ and R¹¹ are hydrogen. In certainembodiments, R¹² and R¹³ are C₁₋₆alkyl, metal cation, or C₁₋₆aralkyl. Incertain preferred embodiments, R¹² and R¹³ are selected from benzyl,tert-butyl, and sodium cation. In more preferred embodiments, both R¹²and R¹³ are benzyl or tert-butyl. In the most preferred embodiment, atleast one of R¹² and R¹³ is a sodium cation.

In certain embodiments, specifically excluded compounds are disclosed inU.S. Pat. No. 6,831,099, the disclosure of which is incorporated hereinby reference in its entirety.

One aspect of the invention relates to a medical device includingcomposition disclosed herein that include an inhibitor having astructure of any one of formulae I, II, III, or V. In one embodiment,the composition is incorporated within a medical device. In certainembodiments, the medical device is a gel comprising a polymer matrix orceramic matrix and an inhibitor. Said polymer can be either naturallyoccurring or synthetic. In another embodiment, said gel serves as a drugdepot, an adhesive, a suture, a barrier or a sealant.

Another aspect of the invention relates to a medical device comprising asubstrate having a surface onto which an inhibitor having a structure ofany one of formulae I, II, III, or V is disposed. In one embodiment, theinhibitor is directly disposed on a medical device. In anotherembodiment, a coating is so disposed, the coating comprising a polymermatrix or ceramic matrix with an inhibitor having a structure of any oneof formulae I, II, III, or V dispersed or dissolved therein.

In one embodiment, the medical device is a coronary, vascular,peripheral, or biliary stent. More particularly, the stent of thepresent invention is an expandable stent. When coated with a matrixcontaining an inhibitor having a structure any one of formulae I, II,III, or V, the matrix is flexible to accommodate compressed and expandedstates of such an expandable stent. In another embodiment of thisinvention, the stent has at least a portion which is insertable orimplantable into the body of a patient, wherein the portion has asurface which is adapted for exposure to body tissue and wherein atleast a part of the surface is coated with an inhibitor having astructure of any one of formulae I, II, III, or V, or a coatingcomprising a matrix having an inhibitor having a structure of any one offormulae I, II, III, or V is dispersed or dissolved therein. An exampleof a suitable stent is disclosed in U.S. Pat. No. 4,733,665, which isincorporated herein by reference in its entirety.

In another embodiment, the medical device of the present invention is asurgical implement such as a vascular implant, an intraluminal device,surgical sealant or a vascular support. More particularly, the medicaldevice of the present invention is a catheter, an implantable vascularaccess port, a central venous catheter, an arterial catheter, a vasculargraft, an intraaortic balloon pump, a suture, a ventricular assist pump,a drug-eluting barrier, an adhesive, a vascular wrap, anextra/perivascular support, a blood filter, or a filter adapted fordeployment in a blood vessel, coated with an inhibitor having astructure of any one of formulae I, II, III, or V either directly or bya matrix containing an inhibitor having a structure of any one offormulae I, II, III, or V.

In certain embodiments, the intraluminal medical device is coated withan inhibitor having a structure of any one of formulae I, II, III, or Vor a coating comprising biologically tolerated matrix and an inhibitorhaving a structure of any one of formulae I, II, III, or V dispersed inthe polymer, said device having an interior surface and an exteriorsurface, having the coating applied to at least a part of the interiorsurface, the exterior surface, or both.

In certain embodiments, the medical device may be useful to preventrestenosis after angioplasty. The medical device may also be useful forthe treatment of various diseases and conditions by providing localizedadministration of an inhibitor having a structure of any one of formulaeI, II, III, or V. Such diseases and conditions include restenosis,inflammation, rheumatoid arthritis, tissue injury due to inflammation,hyperproliferative diseases, severe or arthritic psoriasis,muscle-wasting diseases, chronic infectious diseases, abnormal immuneresponse, conditions involving vulnerable plaques, injuries related toischemic conditions, and viral infection and proliferation. Examples ofdiseases and conditions that are subject to a treatment including thedrug coated medical devices of the present invention includeatherosclerosis, acute coronary syndrome, Alzheimer's disease, cancer,fever, muscle disuse (atrophy), denervation, vascular occlusions,stroke, HIV infection, nerve injury, renal failure associated withacidosis, and hepatic failure. See, e.g., Goldberg, U.S. Pat. No.5,340,736.

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain, includinghaloalkyl groups such as trifluoromethyl and 2,2,2-tirfluoroethyl, etc.C₀alkyl indicates a hydrogen where the group is in a terminal position,a bond if internal. The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl”refer to substituted or unsubstituted unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C₁₋₆alkoxyalkyl” refers to a C₁₋₆alkyl group substituted withan alkoxy group, thereby forming an ether.

The term “C₁₋₆aralkyl”, as used herein, refers to a C₁₋₆alkyl groupsubstituted with an aryl group.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by the general formulae:

wherein R⁹, R¹⁰ and R^(10′) each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger from 1 to 8. In preferred embodiments, only one of R⁹ or R¹⁰ canbe a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form animide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionallyR^(10′)) each independently represent a hydrogen, an alkyl, an alkenyl,or —(CH₂)_(m)—R⁸. In certain embodiments, an amino group is basic,meaning it has a pK_(a)≧7.00. The protonated forms of these functionalgroups have pK_(a)s above 7.00.

The terms “amide” and “amido” are art-recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

wherein R⁹, R¹⁰ are as defined above. Preferred embodiments of the amidewill not include imides which may be unstable.

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsubstituted or unsubstituted single-ring aromatic groups in which eachatom of the ring is carbon. The term “aryl” also includes polycyclicring systems having two or more cyclic rings in which two or morecarbons are common to two adjoining rings wherein at least one of therings is aromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline,and the like.

The terms “carbocycle” and “carbocyclyl”, as used herein, refer to anon-aromatic substituted or unsubstituted ring in which each atom of thering is carbon. The terms “carbocycle” and “carbocyclyl” also includepolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is carbocyclic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R^(11′) represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above.Where X is an oxygen and R¹¹ or R^(11′) is not hydrogen, the formularepresents an “ester”. Where X is an oxygen, and R¹¹ is a hydrogen, theformula represents a “carboxylic acid”.

As used herein, “enzyme” can be any partially or wholly proteinaceousmolecule which carries out a chemical reaction in a catalytic manner.Such enzymes can be native enzymes, fusion enzymes, proenzymes,apoenzymes, denatured enzymes, farnesylated enzymes, ubiquitinatedenzymes, fatty acylated enzymes, gerangeranylated enzymes, GPI-linkedenzymes, lipid-linked enzymes, prenylated enzymes, naturally-occurringor artificially-generated mutant enzymes, enzymes with side chain orbackbone modifications, enzymes having leader sequences, and enzymescomplexed with non-proteinaceous material, such as proteoglycans,proteoliposomes. Enzymes can be made by any means, including naturalexpression, promoted expression, cloning, various solution-based andsolid-based peptide syntheses, and similar methods known to those ofskill in the art.

The term “C₁₋₆heteroaralkyl”, as used herein, refers to a C₁₋₆alkylgroup substituted with a heteroaryl group.

The terms “heteroaryl” includes substituted or unsubstituted aromatic 5-to 7-membered ring structures, more preferably 5- to 6-membered rings,whose ring structures include one to four heteroatoms. The term“heteroaryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is heteroaromatic, e.g., theother cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, forexample, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen,phosphorus, and sulfur.

The terms “heterocyclyl” or “heterocyclic group” refer to substituted orunsubstituted non-aromatic 3- to 10-membered ring structures, morepreferably 3- to 7-membered rings, whose ring structures include one tofour heteroatoms. The term terms “heterocyclyl” or “heterocyclic group”also include polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings wherein atleast one of the rings is heterocyclic, e.g., the other cyclic rings canbe cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heterocyclyl groups include, for example, piperidine,piperazine, pyrrolidine, morpholine, lactones, lactams, and the like.

The term “C₁₋₆hydroxyalkyl” refers to a C₁₋₆alkyl group substituted witha hydroxy group.

As used herein, the term “inhibitor” is meant to describe a compoundthat blocks or reduces an activity of an enzyme (for example, inhibitionof proteolytic cleavage of standard fluorogenic peptide substrates suchas suc-LLVY-AMC (SEQ ID NO: 1), Box-LLR-AMC and Z-LLE-AMC, inhibition ofvarious catalytic activities of the 20S proteasome). An inhibitor canact with competitive, uncompetitive, or noncompetitive inhibition. Aninhibitor can bind reversibly or irreversibly, and therefore the termincludes compounds that are suicide substrates of an enzyme. Aninhibitor can modify one or more sites on or near the active site of theenzyme, or it can cause a conformational change elsewhere on the enzyme.

As used herein, the term “peptide” includes not only standard amidelinkage with standard α-substituents, but commonly utilizedpeptidomimetics, other modified linkages, non-naturally occurring sidechains, and side chain modifications, as detailed below.

The terms “polycyclyl” or “polycyclic” refer to two or more rings (e.g.,cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Each of the rings of thepolycycle can be substituted or unsubstituted.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” encompasses compounds that, under physiologicalconditions, are converted into therapeutically active agents. A commonmethod for making a prodrug is to include selected moieties that arehydrolyzed under physiological conditions to reveal the desiredmolecule. In other embodiments, the prodrug is converted by an enzymaticactivity of the host animal.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include, for example, a halogen, ahydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl,or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, aphosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro,an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or anaromatic or heteroaromatic moiety. It will be understood by thoseskilled in the art that the moieties substituted on the hydrocarbonchain can themselves be substituted, if appropriate.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment, refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “thioether” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In preferred embodiments, the“thioether” is represented by —S-alkyl. Representative thioether groupsinclude methylthio, ethylthio, and the like.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

Selectivity for 20S Proteasome

The enzyme inhibitor analogs and prodrugs disclosed herein are useful inpart because they inhibit the action of the 20S proteasome.Additionally, unlike other 20S proteasome inhibitors, the compoundsdisclosed herein are highly selective toward the 20S proteasome, withrespect to other protease enzymes. That is, the instant compounds showselectivities for the 20S proteasome over other proteases such ascathepsins, calpains, papain, chymotrypsin, trypsin, tripeptidylpepsidase II. The selectivities of the enzyme inhibitors for 20Sproteasome are such that at concentrations below about 50 μM, the enzymeinhibitors show inhibition of the catalytic activity of the 20Sproteasome, while not showing inhibition of the catalytic activity ofother proteases such as cathepsins, calpains, papain, chymotrypsin,trypsin, tripeptidyl pepsidase II. In preferred embodiments, the enzymeinhibitors show inhibition of the catalytic activity of the 20Sproteasome at concentrations below about 10 μM, while not showinginhibition of the catalytic activity of other proteases at theseconcentrations. In even more preferred embodiments, the enzymeinhibitors show inhibition of the catalytic activity of the 20Sproteasome at concentrations below about 1 μM, while not showinginhibition of the catalytic activity of other proteases at theseconcentrations. Enzyme kinetic assays are disclosed in U.S. applicationSer. No. 09/569,748, Example 2 and Stein et al., Biochem. (1996), 35,3899-3908.

Selectivity for Chymotrypsin-Like Activity

Particular embodiments of the enzyme inhibiting analog and prodrugcompounds described herein are further useful because they canefficiently and selectively inhibit the chymotrypsin-like activity ofthe 20S proteasome, as compared to the trypsin-like, and PGPHactivities. The chymotrypsin-like activity of 20S proteasome ischaracterized by cleavage of peptides in the immediate vicinity of largehydrophobic residues. In particular, the chymotrypsin-like activity ofNtn hydrolases can be determined by cleavage of a standard substrate.Examples of such substrates are known in the art. For example, aleucylleucylvalinyltyrosine derivative can be used. Enzyme kineticassays are disclosed in U.S. application Ser. No. 09/569,748, Example 2and Stein et al., Biochem. (1996), 35, 3899-3908.

Uses of Enzyme Inhibitors

The biological consequences of proteasome inhibition are numerous. Atthe cellular level, the accumulation of polyubiquitinated proteins, cellmorphological changes, and apoptosis have been reported upon treatmentof cells with various proteasome inhibitors. Proteasome inhibition hasalso been suggested as a possible antitumor therapeutic strategy. Thefact that epoxomicin was initially identified in a screen for antitumorcompounds validates the proteasome as an antitumor chemotherapeutictarget. Accordingly, these compounds are useful for treating cancer.Proteasome inhibition has also been associated with inhibition of NF-κBactivation and stabilization of p53 levels. Thus, compounds of theinvention may also be used to inhibit NF-κB activation, and stabilizep53 levels in cell culture. Since NF-κB is a key regulator ofinflammation, it is an attractive target for anti-inflammatorytherapeutic intervention. Thus, compounds of the invention may be usefulfor the treatment of conditions associated with chronic inflammation,including, but not limited to COPD, psoriasis, bronchitis, emphysema,and cystic fibrosis.

The disclosed compounds can be used to treat conditions mediateddirectly by the proteolytic function of the proteasome such as musclewasting, or mediated indirectly via proteins which are processed by theproteasome such as NF-κB. The proteasome participates in the rapidelimination and post-translational processing of proteins (e.g.,enzymes) involved in cellular regulation (e.g., cell cycle, genetranscription, and metabolic pathways), intercellular communication, andthe immune response (e.g., antigen presentation). Specific examplesdiscussed below include β-amyloid protein and regulatory proteins suchas cyclins and transcription factor NF-κB.

Another embodiment of the invention is the use of the compoundsdisclosed herein for the treatment of neurodegenerative diseases andconditions, including, but not limited to, stroke, ischemic damage tothe nervous system, neural trauma (e.g., percussive brain damage, spinalcord injury, and traumatic damage to the nervous system), multiplesclerosis and other immune-mediated neuropathies (e.g., Guillain-Barresyndrome and its variants, acute motor axonal neuropathy, acuteinflammatory demyelinating polyneuropathy, and Fisher Syndrome),HIV/AIDs dementia complex, axonomy, diabetic neuropathy, Parkinson'sdisease, Huntington's disease, multiple sclerosis, bacterial, parasitic,fungal, and viral meningitis, encephalitis, vascular dementia,multi-infarct dementia, Lewy body dementia, frontal lobe dementia suchas Pick's disease, subcortical dementias (such as Huntington orprogressive supranuclear palsy), focal cortical atrophy syndromes (suchas primary aphasia), metabolic-toxic dementias (such as chronichypothyroidism or B12 deficiency), and dementias caused by infections(such as syphilis or chronic meningitis).

Alzheimer's disease is characterized by extracellular deposits ofβ-amyloid protein (β-AP) in senile plaques and cerebral vessels. β-AP isa peptide fragment of 39 to 42 amino acids derived from an amyloidprotein precursor (APP). At least three isoforms of APP are known (695,751, and 770 amino acids). Alternative splicing of mRNA generates theisoforms; normal processing affects a portion of the β-AP sequence,thereby preventing the generation of β-AP. It is believed that abnormalprotein processing by the proteasome contributes to the abundance ofβ-AP in the Alzheimer brain. The APP-processing enzyme in rats containsabout ten different subunits (22 kDa-32 kDa). The 25 kDa subunit has anN-terminal sequence of X-Gln-Asn-Pro-Met-X-Thr-Gly-Thr-Ser (SEQ ID NO:2), which is identical to the β-subunit of human macropain (Kojima, S.et al., Fed. Eur. Biochem. Soc., (1992) 304:57-60). The APP-processingenzyme cleaves at the Gln¹⁵-Lys¹⁶ bond; in the presence of calcium ion,the enzyme also cleaves at the Met-¹-Asp¹ bond, and the Asp¹-Ala² bondsto release the extracellular domain of β-AP.

One embodiment, therefore, is a method of treating Alzheimer's disease,including administering to a subject an effective amount of a compound(e.g., pharmaceutical composition) disclosed herein. Such treatmentincludes reducing the rate of β-AP processing, reducing the rate of β-APplaque formation, reducing the rate of β-AP generation, and reducing theclinical signs of Alzheimer's disease.

Other embodiments of the invention relate to cachexia and muscle-wastingdiseases. The proteasome degrades many proteins in maturingreticulocytes and growing fibroblasts. In cells deprived of insulin orserum, the rate of proteolysis nearly doubles Inhibiting the proteasomereduces proteolysis, thereby reducing both muscle protein loss and thenitrogenous load on kidneys or liver. Inhibitors of the invention areuseful for treating conditions such as cancer, chronic infectiousdiseases, fever, muscle disuse (atrophy) and denervation, nerve injury,fasting, renal failure associated with acidosis, diabetes, and hepaticfailure. See, e.g., Goldberg, U.S. Pat. No. 5,340,736. Embodiments ofthe invention therefore encompass methods for: reducing the rate ofmuscle protein degradation in a cell; reducing the rate of intracellularprotein degradation; reducing the rate of degradation of p53 protein ina cell; and inhibiting the growth of p53-related cancers. Each of thesemethods includes contacting a cell (in vivo or in vitro, e.g., a musclein a subject) with an effective amount of a compound (e.g.,pharmaceutical composition) disclosed herein.

Another protein processed by the proteasome is NF-κB, a member of theRel protein family. The Rel family of transcriptional activator proteinscan be divided into two groups. The first group requires proteolyticprocessing, and includes p50 (NF-κB1, 105 kDa) and p52 (NF-κ2, 100 kDa).The second group does not require proteolytic processing, and includesp65 (RelA, Rel (c-Rel), and RelB). Both homo- and heterodimers can beformed by Rel family members; NF-κB, for example, is a p50-p65heterodimer. After phosphorylation and ubiquitination of IκB and p105,the two proteins are degraded and processed, respectively, to produceactive NF-κB which translocates from the cytoplasm to the nucleus.Ubiquitinated p105 is also processed by purified proteasomes (Palombellaet al., Cell (1994) 78:773-785). Active NF-κB forms a stereospecificenhancer complex with other transcriptional activators and, e.g., HMGI(Y), inducing selective expression of a particular gene.

NF-κB regulates genes involved in the immune and inflammatory response,and mitotic events. For example, NF-κB is required for the expression ofthe immunoglobulin light chain κ gene, the IL-2 receptor α-chain gene,the class I major histocompatibility complex gene, and a number ofcytokine genes encoding, for example, IL-2, IL-6, granulocytecolony-stimulating factor, and IFN-β (Palombella et al., Cell (1994)78:773-785). Some embodiments of the invention include methods ofaffecting the level of expression of IL-2, MHC-I, IL-6, TNFα, IFN-β orany of the other previously-mentioned proteins, each method includingadministering to a subject an effective amount of a compound disclosedherein. Complexes including p50 are rapid mediators of acuteinflammatory and immune responses (Thanos, D. and Maniatis, T., Cell(1995) 80:529-532).

NF-κB also participates in the expression of the cell adhesion genesthat encode E-selectin, P-selectin, ICAM, and VCAM-1 (Collins, T., Lab.Invest. (1993) 68:499-508). One embodiment of the invention is a methodfor inhibiting cell adhesion (e.g., cell adhesion mediated byE-selectin, P-selectin, ICAM, or VCAM-1), including contacting a cellwith (or administering to a subject) an effective amount of a compound(or a pharmaceutical composition) disclosed herein.

NF-κB also binds specifically to the HIV-enhancer/promoter. Whencompared to the Nef of mac239, the HIV regulatory protein Nef of pbj14differs by two amino acids in the region which controls protein kinasebinding. It is believed that the protein kinase signals thephosphorylation of IκB, triggering IκB degradation through theubiquitin-proteasome pathway. After degradation, NF-κB is released intothe nucleus, thus enhancing the transcription of HIV (Cohen, J.,Science, (1995) 267:960). Two embodiments of the invention are a methodfor inhibiting or reducing HIV infection in a subject, and a method fordecreasing the level of viral gene expression, each method includingadministering to the subject an effective amount of a compound disclosedherein.

Overproduction of lipopolysaccharide (LPS)-induced cytokines such asTNFα is considered to be central to the processes associated with septicshock. Furthermore, it is generally accepted that the first step in theactivation of cells by LPS is the binding of LPS to specific membranereceptors. The α- and β-subunits of the 20S proteasome complex have beenidentified as LPS-binding proteins, suggesting that the LPS-inducedsignal transduction may be an important therapeutic target in thetreatment or prevention of sepsis (Qureshi, N. et al., J. Immun. (2003)171: 1515-1525). Therefore, in certain embodiments, compounds of theinvention may be used for the inhibition of TNFα to prevent and/or treatseptic shock.

Intracellular proteolysis generates small peptides for presentation toT-lymphocytes to induce MHC class I-mediated immune responses. Theimmune system screens for autologous cells that are virally infected orhave undergone oncogenic transformation. One embodiment is a method forinhibiting antigen presentation in a cell, including exposing the cellto a compound described herein. A compound of the invention may be usedto treat immune-related conditions such as allergy, asthma, organ/tissuerejection (graft-versus-host disease), and auto-immune diseases,including, but not limited to, lupus, rheumatoid arthritis, psoriasis,multiple sclerosis, and inflammatory bowel diseases (such as ulcerativecolitis and Crohn's disease). Thus, a further embodiment is a method forsuppressing the immune system of a subject (e.g., inhibiting transplantrejection, allergies, and asthma), including administering to thesubject an effective amount of a compound described herein.

Another further embodiment is a method for altering the repertoire ofantigenic peptides produced by the proteasome or other Ntn withmulticatalytic activity. For example, if the PGPH activity of 20Sproteasome is selectively inhibited, a different set of antigenicpeptides will be produced by the proteasome and presented in MHCmolecules on the surfaces of cells than would be produced and presentedeither without any enzyme inhibition, or with, for example, selectiveinhibition of chymotrypsin-like activity of the proteasome.

Certain proteasome inhibitors block both degradation and processing ofubiquitinated NF-κB in vitro and in vivo. Proteasome inhibitors alsoblock IκB-α degradation and NF-κB activation (Palombella, et al. Cell(1994) 78:773-785; and Traenckner, et al., EMBO J. (1994) 13:5433-5441).One embodiment of the invention is a method for inhibiting Ii(B-αdegradation, including contacting the cell with a compound describedherein. A further embodiment is a method for reducing the cellularcontent of NF-κB in a cell, muscle, organ, or subject, includingcontacting the cell, muscle, organ, or subject with a compound describedherein.

Other eukaryotic transcription factors that require proteolyticprocessing include the general transcription factor TFIIA, herpessimplex virus VP16 accessory protein (host cell factor), virus-inducibleIFN regulatory factor 2 protein, and the membrane-bound sterolregulatory element-binding protein 1.

Other embodiments of the invention are methods for affectingcyclin-dependent eukaryotic cell cycles, including exposing a cell (invitro or in vivo) to a compound disclosed herein. Cyclins are proteinsinvolved in cell cycle control. The proteasome participates in thedegradation of cyclins. Examples of cyclins include mitotic cyclins, G1cyclins, and cyclin B. Degradation of cyclins enables a cell to exit onecell cycle stage (e.g., mitosis) and enter another (e.g., division). Itis believed all cyclins are associated with p34.sup.cdc2 protein kinaseor related kinases. The proteolysis targeting signal is localized toamino acids 42-RAALGNISEN-50 (SEQ ID NO: 3) (destruction box). There isevidence that cyclin is converted to a form vulnerable to a ubiquitinligase or that a cyclin-specific ligase is activated during mitosis(Ciechanover, A., Cell, (1994) 79:13-21) Inhibition of the proteasomeinhibits cyclin degradation, and therefore inhibits cell proliferation,for example, in cyclin-related cancers (Kumatori et al., Proc. Natl.Acad. Sci. USA (1990) 87:7071-7075). One embodiment of the invention isa method for treating a proliferative disease in a subject (e.g.,cancer, psoriasis, or restenosis), including administering to thesubject an effective amount of a compound disclosed herein. Theinvention also encompasses a method for treating cyclin-relatedinflammation in a subject, including administering to a subject atherapeutically effective amount of a compound described herein.

Additional embodiments are methods for affecting theproteasome-dependent regulation of oncoproteins and methods of treatingor inhibiting cancer growth, each method including exposing a cell (invivo, e.g., in a subject, or in vitro) to a compound disclosed herein.HPV-16 and HPV-18-derived E6 proteins stimulate ATP- andubiquitin-dependent conjugation and degradation of p53 in crudereticulocyte lysates. The recessive oncogene p53 has been shown toaccumulate at the nonpermissive temperature in a cell line with amutated thermolabile E1. Elevated levels of p53 may lead to apoptosis.Examples of proto-oncoproteins degraded by the ubiquitin system includec-Mos, c-Fos, and c-Jun. One embodiment is a method for treatingp53-related apoptosis, including administering to a subject an effectiveamount of a compound disclosed herein.

In another embodiment, the disclosed compounds are useful for thetreatment of a parasitic infection, such as infections caused byprotozoan parasites. The proteasome of these parasites is considered tobe involved primarily in cell differentiation and replication activities(Paugam et al., Trends Parasitol. 2003, 19(2): 55-59). Furthermore,entamoeba species have been shown to lose encystation capacity whenexposed to proteasome inhibitors (Gonzales, et al., Arch. Med. Res.1997, 28, Spec No: 139-140). In certain such embodiments, the disclosedcompounds are useful for the treatment of parasitic infections in humanscaused by a protozoan parasite selected from Plasmodium sps. (includingP. falciparum, P. vivax, P. malariae, and P. ovale, which causemalaria), Trypanosoma sps. (including T. cruzi, which causes Chagas'disease, and T. brucei which causes African sleeping sickness),Leishmania sps. (including L. amazonensis, L. donovani, L. infantum, L.mexicana, etc.), Pneumocystis carinii (a protozoan known to causepneumonia in AIDS and other immunosuppressed patients), Toxoplasmagondii, Entamoeba histolytica, Entamoeba invadens, and Giardia lamblia.In certain embodiments, the disclosed compounds are useful for thetreatment of parasitic infections in animals and livestock caused by aprotozoan parasite selected from Plasmodium hermani, Cryptosporidiumsps., Echinococcus granulosus, Eimeria tenella, Sarcocystis neurona, andNeurospora crassa. Other compounds useful as proteasome inhibitors inthe treatment of parasitic diseases are described in WO 98/10779, whichis incorporated herein in its entirety.

In certain embodiments, the disclosed compounds inhibit proteasomeactivity irreversibly in a parasite. Such irreversible inhibition hasbeen shown to induce shutdown in enzyme activity without recovery in redblood cells and white blood cells. In certain such embodiments, the longhalf-life of blood cells may provide prolonged protection with regard totherapy against recurring exposures to parasites. In certainembodiments, the long half-life of blood cells may provide prolongedprotection with regard to chemoprophylaxis against future infection.

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed compounds may be useful in the treatment and/or prevention ofdiseases associated with bone loss, such as osteroporosis.

Bone tissue is an excellent source for factors which have the capacityfor stimulating bone cells. Thus, extracts of bovine bone tissue containnot only structural proteins which are responsible for maintaining thestructural integrity of bone, but also biologically active bone growthfactors which can stimulate bone cells to proliferate. Among theselatter factors are a recently described family of proteins called bonemorphogenetic proteins (BMPs). All of these growth factors have effectson other types of cells, as well as on bone cells, including Hardy, M.H., et al., Trans Genet (1992) 8:55-61 describes evidence that bonemorphogenetic proteins (BMPs), are differentially expressed in hairfollicles during development. Harris, S. E., et al., J Bone Miner Res(1994) 9:855-863 describes the effects of TGFβ on expression of BMP-2and other substances in bone cells. BMP-2 expression in mature folliclesalso occurs during maturation and after the period of cell proliferation(Hardy, et al. (1992, supra). Thus, compounds of the invention may alsobe useful for hair follicle growth stimulation.

Finally, the disclosed compounds are also useful as diagnostic agents(e.g., in diagnostic kits or for use in clinical laboratories) forscreening for proteins (e.g., enzymes, transcription factors) processedby Ntn hydrolases, including the proteasome. The disclosed compounds arealso useful as research reagents for specifically binding the X/MB1subunit or α-chain and inhibiting the proteolytic activities associatedwith it. For example, the activity of (and specific inhibitors of) othersubunits of the proteasome can be determined

Most cellular proteins are subject to proteolytic processing duringmaturation or activation. Enzyme inhibitors disclosed herein can be usedto determine whether a cellular, developmental, or physiological processor output is regulated by the proteolytic activity of a particular Ntnhydrolase. One such method includes obtaining an organism, an intactcell preparation, or a cell extract; exposing the organism, cellpreparation, or cell extract to a compound disclosed herein; exposingthe compound-exposed organism, cell preparation, or cell extract to asignal, and monitoring the process or output. The high selectivity ofthe compounds disclosed herein permits rapid and accurate elimination orimplication of the Ntn (for example, the 20S proteasome) in a givencellular, developmental, or physiological process.

Administration

Compounds prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means, and ifdesired, the active ingredient may be mixed with any conventionaladditive or excipient, such as a binder, a disintegrating agent, alubricant, a corrigent, a solubilizing agent, a suspension aid, anemulsifying agent, a coating agent, a cyclodextrin, and/or a buffer.Although the dosage will vary depending on the symptoms, age and bodyweight of the patient, the nature and severity of the disorder to betreated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions of the present invention arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, laurylsulphonate salts, and amino acidsalts, and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66: 1-19.)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acomposition may also be administered as a bolus, electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient ismixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, cyclodextrins, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation, orsolid particles containing the composition. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the inhibitor(s) ina polymer matrix or gel.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The concentration of a disclosed compound in a pharmaceuticallyacceptable mixture will vary depending on several factors, including thedosage of the compound to be administered, the pharmacokineticcharacteristics of the compound(s) employed, and the route ofadministration. In general, the compositions of this invention may beprovided in an aqueous solution containing about 0.1-10% w/v of acompound disclosed herein, among other substances, for parenteraladministration. Typical dose ranges are from about 0.01 to about 50mg/kg of body weight per day, given in 1-4 divided doses. Each divideddose may contain the same or different compounds of the invention. Thedosage will be an effective amount depending on several factorsincluding the overall health of a patient, and the formulation and routeof administration of the selected compound(s).

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteaseinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential, or separate dosing of the individualcomponents of the treatment.

In certain embodiments, a compound of the invention is conjointlyadministered with a chemotherapeutic. Suitable chemotherapeutics mayinclude, natural products such as vinca alkaloids (i.e. vinblastine,vincristine, and vinorelbine), paclitaxel, epidipodophyllotoxins (i.e.etoposide, teniposide), antibiotics (dactinomycin (actinomycin D)daunorubicin, doxorubicin and idarubicin), anthracyclines, mitoxantrone,bleomycins, plicamycin (mithramycin) and mitomycin, enzymes(L-asparaginase which systemically metabolizes L-asparagine and deprivescells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates(busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin),trazenes—dacarbazinine (DTIC); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate), pyrimidineanalogs (fluorouracil, floxuridine, and cytarabine), purine analogs andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane,and letrozole); and platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;hormones (i.e. estrogen) and hormone agonists such as leutinizinghormone releasing hormone (LHRH) agonists (goserelin, leuprolide andtriptorelin). Other chemotherapeutic agents may include mechlorethamine,camptothecin, ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine,or any analog or derivative variant of the foregoing.

In certain embodiments, a compound of the invention is conjointlyadministered with a steroid. Suitable steroids may include, but are notlimited to, 21-acetoxypregnenolone, alclometasone, algestone,amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone,clobetasol, clocortolone, cloprednol, corticosterone, cortisone,cortivazol, deflazacort, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difuprednate, enoxolone, fluazacort,flucloronide, flumethasone, flunisolide, fluocinolone acetonide,fluocinonide, fluocortin butyl, fluocortolone, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandrenolide, fluticasone propionate, formocortal, halcinonide,halobetasol propionate, halometasone, hydrocortisone, loteprednoletabonate, mazipredone, medrysone, meprednisone, methylprednisolone,mometasone furoate, paramethasone, prednicarbate, prednisolone,prednisolone 25-diethylaminoacetate, prednisolone sodium phosphate,prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide,triamcinolone hexacetonide, and salts and/or derivatives thereof.

In certain embodiments, a compound of the invention is conjointlyadministered with an immunotherapeutic agent. Suitable immunotherapeuticagents may include, but are not limited to, cyclosporine, thalidomide,and monoclonal antibodies. The monoclonal antibodies can be either nakedor conjugated such as rituximab, tositumomab, alemtuzumab, epratuzumab,ibritumomab tiuxetan, gemtuzumab ozogamicin, bevacizumab, cetuximab,erlotinib and trastuzumab.

EXEMPLIFICATION

Synthesis of (B)

To a solution of NBoc leucine (50.0 mmol, 11.56 g) and phenylalaninemethyl ester (50.0 mmol, 10.78 g) in 500 mL of DMF was added HOBT (10.81g, 80 0 mmol) and DIEA (200.0 mmol, 25.85 g, 35 mL). The mixture wascooled to 0° C. in an ice-water bath and BOP (80.0 mmol, 35.38 g) wasadded in several portions over five minutes. The reaction was placedunder an atmosphere of argon and stirred overnight. The reaction wasdiluted with brine (1000 mL) and extracted with EtOAc (5×200 mL). Theorganic layers were combined and washed with water (10×100 mL) and brine(2×150 mL) and dried over MgSO₄. The MgSO₄ was removed by filtration andthe volatiles removed under reduced pressure to give (A) (18.17 g). To a50 mL 0° C. cooled solution of 80% TFA/DCM was added BocNHLeuPheOMe(45.86 mmol, 18.0 g). The solution was stirred and allowed to warm toroom temperature over 2 hr. The volatiles were removed under reducedpressure to give an oil. To this oil was added BocNHhPhe (45.86 mmol,12.81 g), DMF (500 mL), HOBT (73.37 mmol, 9.91 g) and DIEA (183.44 mmol,23.70 g, 32.0 mL). The mixture was cooled to 0° C. in an ice-water bathand BOP (73.37 mmol, 32.45 g) was added in several portions over fiveminutes. The reaction was placed under argon and allowed to warm to roomtemperature overnight. The reaction was diluted with H₂O (1500 mL) andextracted with DCM (5×300 mL). The organic layers were combined andwashed with H₂O (6×300 mL) and brine (1×300 mL) and dried over MgSO₄.The MgSO₄ was removed by filtration and the volatiles removed underreduced pressure to give a yellow solid. To the solid was added 200 mLof 95% EtOH and the mixture was heated to 65° C. to dissolve all of thesolids. The solution was then added to 1000 mL of chilled H₂O and theresulting precipitate collected to give (B) (21.59 g).

Synthesis of (C)

Compound (B) (1.47 mmol, 0.81 g) was mixed with TFA/DCM (80%) andstirred at room temperature for 1 hr, at which time the mixture wasconcentrated and placed under high vacuum for 2 hr giving the TFA saltof the tri-peptide amine (Q). To a 0° C. solution of the TFA salt (1.47mmol) in DMF (10 mL) was added DIEA (4.4 mmol, 0.77 mL) followed bybenzyloxyacetyl chloride (2.21 mmol, 0.343 mL). The reaction was allowedto warm to RT while stirring for 2 hr under an atmosphere of nitrogen.The mixture was diluted with brine (15 mL) and extracted with EtOAc(3×15 mL). The organic layers were combined, washed with H₂O (2×15 mL),brine (1×15 mL) and dried over Na₂SO₄. The Na₂SO₄ was removed byfiltration and the volatiles removed under reduced pressure. The crudematerial was purified by flash chromatography to afford (C) (0.83 g).

Synthesis of (D)

To a slurry of (C) (1.38 mmol, 0.830 g) in 28 mL of 3:1 MeOH/H₂O cooledto 0° C. was added LiOH (13.8 mmol, 0.331 g). After 18 hr at 5° C., thereaction was quenched with 20 mL sat. NH₄Cl and diluted further with 150mL H₂O. The pH of the reaction mixture was adjusted to 2 with 1N HCl andthe solids were collected by filtration to give (D) (0.900 g).

Synthesis of (E)

Compound (D) (0.17 mmol, 0.10 g) was dissolved in MeOH (10 mL) and Pd—C(5%, 0.08 g) was added, and the reaction mixture stirred under 1atmosphere of H₂ at room temperature for 2 hr. The mixture was thenpurged with argon, filtered through Celite, and concentrated to provide(E).

Synthesis of Compound 1

To a stirred solution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9,2283-88] (0.164 mmol) in DMF (2 mL) was added (E) (0.17 mmol), DIEA(0.652 mmol, 0.114 mL), and HOBT (0.266 mmol, 0.036 g). The mixture wascooled to 0° C. in an ice bath and BOP (0.262 mmol, 0.116 g) was addedin several portions. The mixture was stirred at 5° C. under anatmosphere of argon overnight. The reaction was diluted with brine (15mL) and extracted with EtOAc. The organic layer was washed with water,sat. NaHCO₃, and brine and dried over anhydrous MgSO₄. The MgSO₄ wasremoved by filtration and the volatiles removed under reduced pressure.The crude material was purified by preparative HPLC to afford compound 1(IC₅₀ 20S CT-L<50 nM, IC₅₀ Cell-Based CT-L<100 nM)

Synthesis of (G)

Compound (C) (1.0 mmol, 0.601 g) was dissolved in MeOH (25 mL), Pd—C(10%, 600 mg) was added and the reaction mixture was stirred under 1atmosphere of H₂ at room temperature for 48 hr. The mixture was thenpurged with argon, filtered through Celite, and concentrated to provide(G) (600 mg).

Synthesis of (H)

To a 0° C. solution of (G) (1.0 mmol, 0.511 g) in DCM (40 mL) was addedDIEA (2.0 mmol, 0.348 mL) and dibenzylphosphoryl chloride (2.0 mmol,0.593 g) and the mixture was allowed to stir overnight at roomtemperature under an atmosphere of nitrogen. The reaction was thenconcentrated under vacuum and the crude material purified by flashchromatography to afford (H) (0.181 g).

Synthesis of (I)

To a slurry of (H) (0.11 mmol, 0.090 g) in 4 mL of 3:1 MeOH/H₂O cooledto 0° C. was added LiOH (1.6 mmol, 0.4 mL, 4 M aq.). After 45 minutes at5° C., the reaction was quenched with 10 mL sat. NH₄Cl. The pH of thereaction mixture was adjusted to 2 with 1 N HCl and extracted withEtOAc. The organic layer was washed with water and brine and dried overanhydrous MgSO₄. The MgSO₄ was removed by filtration and the volatilesremoved under reduced pressure to give (I).

Synthesis of (J)

To a stirred solution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9,2283-88] (0.082 mmol) in DMF (2 mL) was added (I) (0.082 mmol, 0.062 g),DIEA (0.328 mmol, 0.057 mL) and HOBT (0.133 mmol, 0.018 g). The mixturewas cooled to 0° C. in an ice bath and BOP (0.131 mmol, 0.058 g) wasadded in several portions. The mixture was then stirred at 5° C. underan atmosphere of argon overnight. The reaction was diluted with H₂O (15mL) and (J) was collected by filtration (0.081 g).

Synthesis of Compound 2

To a solution of (J) (0.005 mmol, 0.005 g) in THF (1 mL) was added 4drops of H₂O and 10% Pd/C (5 mg). The mixture was stirred under H₂ atroom temperature for 1 hr, filtered through Celite, and the filtrate wastreated with Na₂CO₃ (0.263 g in 3 mL H₂O). The solids were collected byfiltration and placed under high vacuum to give compound 2 (0.004 g).

Synthesis of (L)

To a solution of (K) (0.19 mmol, 0.10 g) in THF (20 mL) was added KI(0.038 mmol, 0.0063 g), dimethylamine (0.456 mmol, 0.228 mL, 2M in THF)and the mixture was stirred overnight under an atmosphere of nitrogen.The volatiles were removed under reduced pressure and the crude materialwas taken up in EtOAc (15 mL), washed with H₂O (2×10 mL) and brine (2×10mL), and dried over MgSO₄. The MgSO₄ was removed by filtration and thevolatiles removed under reduced pressure to give (L) (0.100 g).

Synthesis of (M)

To a slurry of (L) (0.186 mmol, 0.100 g) in 4 mL of 3:1 MeOH/H₂O cooledto 0° C. was added LiOH (1.86 mmol, 0.045 g). After 12 hr at 5° C., thereaction was quenched with 20 mL sat. NH₄Cl and diluted further with 10mL H₂O. The pH of the reaction mixture was adjusted to 3 with 1 N HCl,extracted with CHCl₃ (3×15 mL), and dried over MgSO₄. The MgSO₄ wasremoved by filtration and the volatiles were removed under reducedpressure to give (M).

Synthesis of Compound 3

To a stirred solution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9,2283-88] (0.082 mmol) in DMF (1 mL) was added (M) (0.021 mmol), DIEA(0.28 mmol, 0.05 mL) and HOBT (0.133 mmol, 0.018 g). The mixture wascooled to 0° C. in an ice bath and BOP (0.131 mmol, 0.058 g) was addedin several portions. The mixture was stirred at 5° C. under anatmosphere of argon overnight. The reaction was then diluted with brine(15 mL) and extracted with EtOAc. The organic layer was washed withwater, sat. NaHCO₃, and brine and dried over anhydrous MgSO₄. The MgSO₄was removed by filtration and the volatiles removed under reducedpressure to afford compound 3 (IC₅₀ 20S CT-L<100 nM; Cell-Based CT-L<100nM).

Synthesis of (N)

Compound (B) (1.80 mmol, 1.0 g) was mixed with TFA/DCM (80%) and wasstirred at room temperature for 1 hr, at which time the mixture wasconcentrated and placed under high vacuum for 2 hr giving the TFA saltof the tri-peptide amine. To a 0° C. solution of the TFA salt (1.80mmol) in DMF (10 mL) was added DIEA (3 6 mmol, 0.7 mL) followed bychloroacetyl chloride (2.7 mmol, 0.215 mL). The reaction was allowed towarm to RT while stirring overnight under an atmosphere of nitrogen. Themixture was then diluted with brine (15 mL) and extracted with EtOAc(3×15 mL). The organic layers were combined, washed with H₂O (2×15 mL)and brine (2×15 mL) and dried over Na₂SO₄. The Na₂SO₄ was removed byfiltration and the volatiles removed under reduced pressure. The crudematerial was suspended in EtOAc and filtered to give (N) (0.640 g)

Synthesis of (O)

To a solution of (N) (0.094 mmol, 0.050 g) in THF (10 mL) was added KI(0.019 mmol, 0.0032 g) and piperidine (0.113 mmol, 0.0096 g) and themixture stirred overnight under an atmosphere of nitrogen. The volatileswere removed under reduced pressure and the crude material taken up inEtOAc (15 mL), washed with H₂O (2×10 mL) and brine (2×10 mL) and driedover MgSO₄. The MgSO₄ was removed by filtration and the volatilesremoved under reduced pressure to give (O).

Synthesis of (P)

To a slurry of (O) (0.094 mmol) in 4 mL of 3:1 MeOH/H₂O cooled to 0° C.was added LiOH (0.94 mmol, 0.023 g). After 12 hr at 5° C. the reactionwas quenched with 20 mL sat. NH₄Cl and diluted further with 10 mL H₂O.The pH of the reaction mixture was adjusted to 3 with 1 N HCl, extractedwith DCM (3×15 mL), and dried over MgSO₄. The MgSO₄ was removed byfiltration and the volatiles were removed under reduced pressure to give(P).

Synthesis of Compound 4

To a stirred solution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9,2283-88] (0.082 mmol) in DMF (2 mL) was added (P) (0.082 mmol, 0.046 g),DIEA (0.328 mmol, 0.057 mL) and HOBT (0.133 mmol, 0.018 g). The mixturewas cooled to 0° C. in an ice bath and BOP (0.131 mmol, 0.058 g) wasadded in several portions. The mixture was stirred at 5° C. under anatmosphere of argon overnight. The reaction was then diluted with H₂O(15 mL) and extracted with EtOAc. The organic layer was washed withwater, sat. NaHCO₃, and brine and dried over anhydrous MgSO₄. The MgSO₄was removed by filtration and the volatiles removed under reducedpressure to give compound 4 (0.034 g) (IC₅₀ 20S CT-L<100 nM; IC₅₀Cell-Based CT-L<100 nM).

Synthesis of (T)

Compound (T) is obtained by essentially following the procedure for theconversion of (E) to 1 but substituting (Q) for (F) andcyclopropylacetic acid for (E).

Synthesis of (U)

Compound (U) is obtained by essentially following the procedure for theconversion of (C) to (D) but substituting (T) for (C).

Synthesis of Compound 5

Compound 5 is obtained by essentially following the procedure for theconversion of (E) to 1 but substituting (U) for (E).

Synthesis of (W)

Compound (V) (0.25 g, 0.39 mmol) was mixed with 12 mL of TFA/DCM (80%)and was stirred at room temperature for 1 hr at which time the mixturewas concentrated and placed under high vacuum for 2 hr giving the TFAsalt of the tri-peptide amine. The crude amine salt was dissolved in 6mL DMF and 2-morpholino acetic acid (0.074 g, 0.507 mmol) was addedfollowed by DIEA (0.504 g, 0.68 mL, 3.90 mmol). The mixture was cooledto 0° C. in an ice bath and PyBOP (0.32 g, 0.62 mmol) was added andstirred under an atmosphere of argon while warming to room temperatureovernight. The mixture was diluted with brine (50 mL) and extracted withEtOAc (5×20 mL). The organic layers were combined, washed with sat.NaHCO₃ (5×15 mL) and brine (1×25 mL) and dried over MgSO₄. The MgSO₄ wasremoved by filtration and the volatiles removed under reduced pressureto give the intermediate ester (0.195 g). To (0.150 g, 0.23 mmol) of theintermediate ester was added 10% Pd/C (0.05 g) followed by 5 mL of 1:1mixture of MeOH and EtOAc and the mixture was placed under an atmosphereof hydrogen. After 2 hr, the contents were filtered through a plug ofCelite and concentrated under vacuum to give (W) (0.12 g).

Synthesis of Compound 6

To a stirred solution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9,2283-88] (1.3 eq., 0.27 mmol, 0.083 mg) in MeCN (5 mL) was added (W) (1eq., 0.17 mmol, 0.10 g), DIEA (10 eq., 1.73 mmol, 0.30 mL) and HOBT (1.6eq., 0.27 mmol, 0.037 mg). The mixture was cooled to 0° C. in an icebath and PyBOP (1.6 eq., 0.27 mmol, 0.14 g) was added in severalportions. The mixture was stirred at 5° C. under an atmosphere of argonovernight after which, the reaction was diluted with sat. NaCl andextracted with EtOAc. The organic layer was washed with water and brineand dried over anhydrous MgSO₄ and concentrated to a paste. The crudematerial was dissolved in a minimum amount of MeOH and slowly added torapidly stirred, 0° C. chilled water (100 mL). Compound 6 was thenisolated by filtration (0.080 g).

Synthesis of (X)

To a solution of NBoc leucine (19.81 g, 85.67 mmol, 1.0 eq) andphenylalanine benzyl ester (25.0 g, 85.67 mmol, 1.0 eq.) in 900 mL ofMeCN was added DIEA (44.29 g, 60 mL, 342.68 mmol, 4.0 eq.) and themixture cooled to 0° C. in an ice bath. To this mixture was added HOBT(18.52 g, 137.08 mmol, 1.6 eq) followed by PyBOP (71.33 g, 137.08 mmol,1.6 eq) which was added in several portions over five minutes. Thereaction was placed under an atmosphere of argon and stirred overnight.The volatiles were removed under reduced pressure and the remainingmaterial was taken up in 500 mL of EtOAc and washed with sat. NaHCO₃,H₂O, and brine and dried over MgSO₄. The MgSO₄ was removed by filtrationand the volatiles removed under reduced pressure to give (X).

Synthesis of (Y)

To a 0° C. cooled solution of 70% TFA/DCM (150 mL) was added (X) (25.0g, 53.35 mmol, 1.0 eq.). The solution was stirred and allowed to warm toroom temperature over 2 hr at which time the mixture was concentratedand placed under high vacuum for 2 hr giving the TFA salt of thedi-peptide amine. To the resulting oil was added BocNHhPhe (14.68 g,53.35 mmol, 1.0 eq.), 550 mL of MeCN, and DIEA (27.58 g, 37.2 mL, 213.4mmol, 4.0 eq.) and the mixture was cooled to 0° C. in an ice bath. Tothe cooled mixture was added HOBT (11.53 g, 85.36 mmol, 1.6 eq.)followed by PyBOP (44.42 g, 85.36 mmol, 1.6 eq.) which was added inseveral portions over five minutes. The reaction was placed under argonand allowed to warm to room temperature overnight at which time a whiteprecipitate had formed. The reaction mixture was cooled and the solidswere collected by filtration and then washed with cold MeCN to give (Y)(24.86 g).

Synthesis of (Z)

Compound (Y) (0.023 mol, 14.5 g) was mixed with TFA/DCM (80%) andstirred at room temperature for 1 hr at which time the mixture wasconcentrated and placed under high vacuum for 2 hr giving the TFA saltof the tri-peptide amine. To a solution of the TFA salt (0.023 mol, 1eq.) in MeCN (120 mL) was added 4-chlorobutyryl chloride (1.2 eq., 0.028mol, 0.32 mL) and DIEA (4 eq., 0.092 mol, 16 mL). The mixture wasstirred at room temperature for 2 hr and then concentrated and purifiedby flash chromatography to afford (Z) (8 g).

Synthesis of (AA)

To a solution of (Z) (1 eq., 0.095 mmol, 60 mg) in dry acetone (8 mL)was added NaI (5 eq., 0.47 mmol, 70.5 mg). The mixture was stirred atreflux under an atmosphere of nitrogen overnight. The reaction mixturewas then concentrated to dryness and the residue taken up in DCM. Theorganic layer was washed with water and brine, dried over anhydrousMgSO₄, and concentrated to give (AA) as a yellow solid (50 mg).

Synthesis of (BB)

To a solution of (AA) (30 mg, 0.041 mmol) in THF (2 mL) was addeddimethylamine (1.2 eq., 0.05 mmol, 2M in THF, 25 μL) and DIEA (1 eq.,0.041 mmol, 7.2 μL). The mixture was stirred at room temperatureovernight and concentrated to dryness. The residue was taken up in ethylacetate and washed with water and brine, dried over anhydrous MgSO₄, andconcentrated to give an oil product. The crude ester was dissolved inMeOH/EtOAc (1:1, 10 mL) and Pd—C (5%, 20 mg) was added and the reactionmixture stirred under 1 atmosphere of H₂ at room temperature for 2 hr.The mixture was then purged with argon, filtered through Celite, andconcentrated to provide (BB) (21 mg).

Synthesis of Compound 7

To a stirred solution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9,2283-88] (1.2 eq., 0.054 mmol) in DMF (3 mL) was added (BB) (1 eq.,0.045 mmol, 21 mg), DIEA (4 eq., 0.18 mmol, 31 μL), and HOBT (1.6 eq.,0.072 mmol, 10 mg). The mixture was cooled to 0° C. in an ice bath andPyBOP (1.6 eq., 0.072 mmol, 37 mg) was added in several portions. Themixture was then stirred at 5° C. under an atmosphere of nitrogenovernight. The reaction was then diluted with sat. NaCl and extractedwith EtOAc. The organic layer was washed with water and brine and driedover anhydrous MgSO₄ and concentrated to an oil that was purified byflash chromatography to afford compound 7 (16.7 mg) (IC₅₀ 20S CT-L<50nM, Cell-Based CT-L<150 nM).

Synthesis of (CC)

Compound (Y) (1.55 g, 0.0023 mol) was dissolved in MeOH/EtOAc (1:1, 40mL) and Pd—C (5%, 500 mg) was added. The mixture was stirred at roomtemperature under hydrogen for 2 hr, and then filtered through Celiteand concentrated to get the carboxylic acid intermediate. To a stirredsolution of (F) [see: Bioorg. Med. Chem. Letter 1999, 9, 2283-88] (1.2eq., 2.55 mmol, 436 mg) in DMF (50 mL) was added the carboxylic acidintermediate (1 eq., 2.12 mmol, 1.24 g), DIEA (4 eq., 8.48 mmol, 1.5 mL)and HOBT (1.6 eq., 3.39 mmol, 458 mg). The mixture was cooled to 0° C.in an ice bath and PyBOP (1.6 eq., 3.39 mmol, 1.76 g) was added inseveral portions. The mixture was stirred at 5° C. under an atmosphereof nitrogen overnight. The reaction was diluted with sat. NaCl andextracted with EtOAc. The organic layer was washed with water and brineand dried over anhydrous MgSO₄ and concentrated to an oil that waspurified by flash chromatography to afford (CC) (356 mg).

Synthesis of Compound 8

Compound (CC) (23.6 mg, 0.034 mmol) was mixed with TFA/DCM (80%) and wasstirred at room temperature for 1 hr at which time the mixture wasconcentrated and placed under high vacuum for 2 hr giving the TFA saltof the tetra-peptide amine. To a DCM solution of the TFA salt was added1,3,5-trimethyl-1-H-pyrazole-4-sulfonyl chloride (1.2 eq., 0.041 mmol,8.5 mg) and TEA (4 eq., 0.136 mmol, 26 μL) and the mixture was stirredat room temperature overnight. The crude mixture was concentrated todryness and the residue was taken up in EtOAc. The organic layer waswashed with water and brine, dried over anhydrous MgSO₄, andconcentrated to an oil that was purified by flash chromatography toafford compound 8 (2 mg) (IC₅₀ 20S CT-L<100 nM, Cell-Based CT-L<100 nM)

Synthesis of Compound 9

Compound (CC) (63.7 mg, 0.092 mmol) was mixed with TFA/DCM (80%) and wasstirred at room temperature for 1 hr at which time the mixture wasconcentrated and placed under high vacuum for 2 hr giving the TFA saltof the tetra-peptide amine. To a DCM solution of the TFA salt was addedphenyl isocyanate (1.5 eq., 0.14 mmol, 15 μL) and DIEA (3 eq., 0.276mmol, 50 μL) and the mixture was stirred at room temperature overnight.The crude mixture was concentrated to dryness and the residue was takenup in EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄, and concentrated to an oil that was purified byflash chromatography to afford compound 9 (2.8 mg).

Synthesis of Compound 10

Compound (CC) (48.5 mg, 0.07 mmol) was mixed with TFA/DCM (80%) and wasstirred at room temperature for 1 hr at which time the mixture wasconcentrated and placed under high vacuum for 2 hr giving the TFA saltof the tetra-peptide amine To a DCM solution of the TFA salt was addedphenyl isothiocyanate (1.5 eq., 0.105 mmol, 20 μL) and DIEA (3 eq., 0.21mmol, 40 μL) and the mixture was stirred at room temperature overnight.The crude mixture was concentrated to dryness and the residue was takenup in EtOAc. The organic layer was washed with water and brine, driedover anhydrous MgSO₄ and concentrated to an oil that was purified byflash chromatography to afford compound 10 (1 mg).

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

1. A method for reducing the rate of p53 protein degradation in one ormore cells, the method comprising contacting one or more cells with acompound having the formula:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein at least one of the one or more cells is a cancer cell.
 3. Themethod of claim 1, wherein said contacting is in vitro.
 4. The method ofclaim 1, wherein said contacting is in vivo.
 5. The method of claim 4,wherein the compound or pharmaceutically acceptable salt thereof is inthe form of a composition comprising the compound or pharmaceuticallyacceptable salt thereof and one or more pharmaceutically acceptablecarriers.
 6. The method of claim 5, wherein each of the one or morepharmaceutically acceptable carriers is independently selected from abinder, a disintegrating agent, a lubricant, a corrigent, a solubilizingagent, a suspension aid, an emulsifying agent, a coating agent, acyclodextrin, and a buffer.
 7. The method of claim 5, wherein each ofthe one or more pharmaceutically acceptable carriers is independentlyselected from: (1) a sugar selected from lactose, glucose, and sucrose;(2) a starch selected from corn starch, potato starch, and substitutedor unsubstituted β-cyclodextrin; (3) cellulose or a derivative selectedfrom sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)an excipient selected from cocoa butter and suppository waxes; (9) anoil selected from peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, and soybean oil; (10) propylene glycol; (11) apolyol selected from glycerin, sorbitol, mannitol, and polyethyleneglycol; (12) an ester selected from ethyl oleate and ethyl laurate; (13)agar; (14) a buffering agent selected from magnesium hydroxide andaluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.
 8. The method ofclaim 5, wherein at least one of the one or more pharmaceuticallyacceptable carriers is a cyclodextrin.
 9. The method of claim 5, whereinat least one of the one or more pharmaceutically acceptable carriers isa substituted or unsubstituted β-cyclodextrin.
 10. The method of claim5, wherein at least one of the one or more pharmaceutically acceptablecarriers is a buffer.
 11. The method of claim 5, wherein the compositionfurther comprises an anti-oxidant.
 12. The method of claim 11, whereinthe anti-oxidant is citric acid.
 13. A method for inhibiting cellproliferation that is associated with a cyclin-related cancer, themethod comprising contacting at least one cell associated with acyclin-related cancer with a compound having the formula:

or a pharmaceutically acceptable salt thereof.
 14. The method of claim13, wherein said contacting is in vitro.
 15. The method of claim 13,wherein said contacting is in vivo.
 16. The method of claim 15, whereinthe compound or pharmaceutically acceptable salt thereof is in the formof a composition comprising the compound or pharmaceutically acceptablesalt thereof and one or more pharmaceutically acceptable carriers. 17.The method of claim 16, wherein each of the one or more pharmaceuticallyacceptable carriers is independently selected from a binder, adisintegrating agent, a lubricant, a corrigent, a solubilizing agent, asuspension aid, an emulsifying agent, a coating agent, a cyclodextrin,and a buffer.
 18. The method of claim 16, wherein each of the one ormore pharmaceutically acceptable carriers is independently selectedfrom: (1) a sugar selected from lactose, glucose, and sucrose; (2) astarch selected from corn starch, potato starch, and substituted orunsubstituted β-cyclodextrin; (3) cellulose or a derivative selectedfrom sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)an excipient selected from cocoa butter and suppository waxes; (9) anoil selected from peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, and soybean oil; (10) propylene glycol; (11) apolyol selected from glycerin, sorbitol, mannitol, and polyethyleneglycol; (12) an ester selected from ethyl oleate and ethyl laurate; (13)agar; (14) a buffering agent selected from magnesium hydroxide andaluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.
 19. The method ofclaim 16, wherein at least one of the one or more pharmaceuticallyacceptable carriers is a cyclodextrin.
 20. The method of claim 16,wherein at least one of the one or more pharmaceutically acceptablecarriers is a substituted or unsubstituted β-cyclodextrin.
 21. Themethod of claim 16, wherein at least one of the one or morepharmaceutically acceptable carriers is a buffer.
 22. The method ofclaim 16, wherein the composition further comprises an anti-oxidant. 23.The method of claim 22, wherein the anti-oxidant is citric acid.
 24. Amethod for inhibiting the growth of a p53-related cancer or acyclin-related cancer in a subject, the method comprising administeringto the subject an effective amount of a compound having the formula:

or a pharmaceutically acceptable salt thereof.
 25. The method of claim24, wherein the compound or pharmaceutically acceptable salt thereof isin the form of a composition comprising the compound or pharmaceuticallyacceptable salt thereof and one or more pharmaceutically acceptablecarriers.
 26. The method of claim 25, wherein each of the one or morepharmaceutically acceptable carriers is independently selected from abinder, a disintegrating agent, a lubricant, a corrigent, a solubilizingagent, a suspension aid, an emulsifying agent, a coating agent, acyclodextrin, and a buffer.
 27. The method of claim 25, wherein each ofthe one or more pharmaceutically acceptable carriers is independentlyselected from: (1) a sugar selected from lactose, glucose, and sucrose;(2) a starch selected from corn starch, potato starch, and substitutedor unsubstituted β-cyclodextrin; (3) cellulose or a derivative selectedfrom sodium carboxymethyl cellulose, ethyl cellulose, and celluloseacetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)an excipient selected from cocoa butter and suppository waxes; (9) anoil selected from peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil, and soybean oil; (10) propylene glycol; (11) apolyol selected from glycerin, sorbitol, mannitol, and polyethyleneglycol; (12) an ester selected from ethyl oleate and ethyl laurate; (13)agar; (14) a buffering agent selected from magnesium hydroxide andaluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations.
 28. The method ofclaim 25, wherein the composition further comprises an anti-oxidant. 29.The method of claim 28, wherein the anti-oxidant is citric acid.
 30. Themethod of claim 24, wherein compound or pharmaceutically acceptable saltthereof is administered by intravenous administration.